EP3197485B1 - Kombiniertes rekombinantes konstrukt aus enterotoxigenem escherichia coli und campylobacter jejuni - Google Patents

Kombiniertes rekombinantes konstrukt aus enterotoxigenem escherichia coli und campylobacter jejuni Download PDF

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EP3197485B1
EP3197485B1 EP15845465.2A EP15845465A EP3197485B1 EP 3197485 B1 EP3197485 B1 EP 3197485B1 EP 15845465 A EP15845465 A EP 15845465A EP 3197485 B1 EP3197485 B1 EP 3197485B1
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etec
subunit
fimbrial
major
construct
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EP3197485A4 (de
EP3197485A1 (de
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Patricia Guerry
Stephen Savarino
Mario Artur Monteiro
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US Department of Navy
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K2039/106—Vibrio; Campylobacter; Not used, see subgroups
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the inventive subject matter relates to a recombinant construct against enterotoxigenic Escherichia coli and Campylobacter jejuni comprising a combined anti-ETEC recombinant polypeptide construct and C . jejuni capsule polysaccharide.
  • ETEC Enterotoxigenic Escherichia coli
  • Shigella Shigella
  • CJ Campylobacter jejuni
  • Both pathogens are a serious health threat to western travelers and young children in resource-limited countries, making them apt target populations for a single or dual pathogen vaccine against ETEC and CJ.
  • No FDA-licensed vaccines are available for either pathogen.
  • ETEC causes an estimated 210 million cases of diarrhea and 380,000 deaths annually among infants and young children. Moreover, ETEC is the most common cause of travelers' diarrhea. ETEC causes diarrhea ranging in severity from mild illness to severe cholera-like purging. There are two major virulence factors, adhesive fimbriae, dubbed colonization factors (CFs), and enterotoxins. Surface-expressed CFs, consisting of complex protein heteropolymers, mediate adherence to the small intestinal epithelium to initiate colonization within this privileged host niche. ETEC produce one or both of two different enterotoxins, a heat-labile (LT) and a heat-stable enterotoxin (STI). LT and STI intoxicate epithelial cells, resulting in fluid and electrolyte secretion and clinical diarrhea. LT is highly immunogenic and a potent adjuvant, while STI is a small, poorly immunogenic peptide.
  • LT heat-labile
  • STI heat-stable enterotoxin
  • CFs and a non-toxic form of the LT have been the focus for several strategies to develop an ETEC vaccine.
  • Such antigens have been used individually or bundled as components of a whole-cell killed vaccine, live vaccines vectored by attenuated ETEC or other enterobacterial species (e.g., Shigella and Vibrio cholerae 01), and purified protein vaccines. None has yet been shown to confer sufficiently high and broad levels of protection.
  • the weight of evidence from clinical trials indicates that anti-LT immunity confers short-term protection against LT-producing ETEC.
  • certain CFs function as protective antigens.
  • ETEC CFs For one, about half of all ETEC express only STI, for which anti-LT immunity is not thought to be effective, thus necessitating anti-CF or anti-bacterial immunity. Also, the diversity of ETEC CFs poses issues for achievement of sufficiently broad coverage with inclusion of a realistic number of CFs.
  • US 2011/0300173 A1 discloses a Campylobacter jejuni construct comprising a polysaccharide from C. jejuni bacterial capsule (e.g., CRM197).
  • WO 2014/077977 A1 discloses enterotoxigenic Escherichia coli (ETEC) constructs.
  • references to methods of treatment in the subsequent paragraphs of this description are to be interpreted as references to the compounds, pharmaceutical compositions and medicaments of the present invention for use in a method for treatment of the human (or animal) body by therapy (or for diagnosis).
  • the invention relates to multi-agent immunogenic construct, comprising a Campylobacter jejuni capsule polysaccharide conjugated to a protein carrier, wherein said protein carrier comprises an enterotoxigenic Escherichia coli (ETEC) recombinant polypeptide construct,
  • ETEC enterotoxigenic Escherichia coli
  • Campylobacger jejuni is associated with induction of Guillain-Barre Syndrome (GBS), a post-infectious polyneuropathy that can result in paralysis.
  • GRS Guillain-Barre Syndrome
  • the association is due to molecular mimicry between the sialic acid containining-outer core of the lipooligosaccharide (LOS) and human gangliosides (5, 6, 89, 91).
  • LOS lipooligosaccharide
  • human gangliosides 5, 6, 89, 91.
  • Use of capsule polysaccharide from C. jejuni can induce an immune response without the possible induction of Guillain-Barre Syndrome.
  • major ETEC fimbrial subunits in order to avoid inadvertent association of subunits, especially in CS6 subunits to each other, can contain an N-terminal deletion of 14 to 18 amino acids.
  • the embodied multipartite construct can contain a deletion of the N-terminal region of one or more fimbrial subunits to avoid undesirable associations with other monomers or multimers and to remove reduce amino acid sequence length between polypeptides to reduce the protease cleavage.
  • DNA encoding the ETEC recombinant polypeptide construct can be used to express a polypeptide for attachment to C. jejuni, and optionnaly Shigella LPS.
  • an object of the invention also includes a multi-agent immunogenic construct of the invention for use in a method of inducing an immune response against C. jejuni strains, wherein said method induces an immune response against one or more enterobacteria selected from the group consisting of C. jejuni strains and Escherichia coli, said method comprising the steps:
  • enterobacteria refers to enterotoxigenic Escherichia coli (ETEC), Campylobacter jejuni or Shigella spp., which include: Shigella dysenteriae, Shigella flexneri, Shigella Boydii or Shigella sonnei.
  • ETEC enterotoxigenic Escherichia coli
  • Salmonella flexneri enterotoxigenic Escherichia coli
  • Shigella Boydii Shigella sonnei.
  • an enterobacteria polysaccharide polymer is a polysaccharide polymer derived from enterobacteria.
  • polysaccharide antigen refers to a capsule polysacchride derived from Campylobacter jejuni ( C.
  • polysaccharide refers to two or more monosaccharide units composing a carbohydrate polymer molecule.
  • a “polysaccharide polymer” refers to two or more polysaccharide molecules connected together.
  • polypeptide refers to a polymer formed of two or more amino acid residues, wherein one or more amino acid residues are naturally occurring amino acids.
  • amino acid sequence refers to the order of the amino acids within a polypeptide.
  • oligomer are polypeptides sequences comprising relatively few amino acids.
  • recombinant polypeptide refers to polypeptides or proteins produced by recombinant DNA techniques, i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide or the desired protein.
  • recombinant construct refers to the DNA encoding the recombinant polypeptide, recombinant polypeptide construct or recombinant protein.
  • donor strand or "donor ⁇ strand” refers to the N-terminal region of an ETEC fimbrial subunit that associates with another ETEC fimbrial subunit in donor strand complementation.
  • immunogenic composition refers to a formulation containing proteins or polypeptides or polysaccharides or polysaccharide polymers that induce a humoral and/or cellular immune response.
  • immuno coverage or “spectrum of coverage” refers to the induction of humoral and/or cellular immune response against specific strains of bacteria under the “coverage.”
  • immunogenogenic fragment refers to a polypeptide containing one or more B- or T-cell epitopes and is of sufficient length to induce an immune response or to be recognized by T- or B-cells.
  • derivative refers to a polypeptide or nucleic acid sequence with at least 80% identity with sequence of the identified gene.
  • identity refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when aligned for maximum correspondence. Where some sequences differ in conservative substitutions, i.e., substitution of residues with identical properties, the the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Percent similarity refers to proportion of identical and similar (conserved change) residues.
  • Fimbriae are defined as projections or filaments on ETEC bacteria and are composed of major subunits, as in the case of CS3 and CS6 fimbriae or major and minor subunits, as in the case of class 5a, 5b and 5c ETEC. "Fibrillae” are narrow projections from a bacteria. CS3 and CS6 fimbriae can also be termed fibrillae due to their narrow characteristic.
  • the term “fimbrial subunit” refers to the proteins that comprise ETEC fimbriae and is used interchangeably with “pilin.” "Pilin”, therefore, can refer to a "major” or “minor” "fimbrial subunit” that comprise ETEC fimbriae.
  • a “minor fimbrial subunit” refers to the adhesin protein at the tip of class 5 ETEC fimbriae and is expressed in stoichiometrically low amounts compared to "major” subunits.
  • the “minor fimbrial subunits” include, but are not limited to, CfaE, CsfD, CsuD, CooD, CosD, CsdD, CsbD and CotD.
  • Major fimbrial subunits refers to the ETEC fimbrial proteins represented in stoichiometrially larger amounts in ETEC fimbriae, compared to “minor fimbrial subunits.”
  • Major fimbrial subunits include the ETEC class 5 proteins: CfaB, CsfA, CsuA2, CsuA1, CooA, CosA, CsdA, CsbA, CotA; the ETEC CS3 proteins: CstH, CstG; and the ETEC CS6 proteins: CssA, and CssB.
  • Campylobacter jejuni The pathogenesis of Campylobacter jejuni remains poorly understood in comparison with ETEC and the organism shares few virulence factors with bettercharacterized pathogens. C. jejuni is unusual, however, among enteric pathogens in that it expresses a polysaccharide capsule (CPS) that is one of its few confirmed virulence factors.
  • CPS polysaccharide capsule
  • a combined ETEC-CJ composition was constructed in order to afford protection against both agents.
  • a recombinant polypeptide construct comprising fimbrial subunits from Class 5 ETEC strains is fused to a capsule polysaccharided from the C. jejuni strain 18-176.
  • one or more recombinant polypeptide ETEC constructs comprising the ETEC fimbrial adhesion, are conjugated to isolated C. jejuni capsule polysaccharide (CPS).
  • C. jejuni capsule polysaccharide C. jejuni capsule polysaccharide
  • One or more of a number of ETEC recombinant constructs can be conjugated to one or more of a number of C. jejuni capsule polysaccharide structures.
  • the ETEC recombinant construct operates both as an immunogen against ETEC and as a protein carrier molecule, presenting the C. jejuni polysaccharide. Examples of ETEC recombinant polypeptides and C. jejuni capsule polysaccharides that can be incorporated into a combined structure are given in the following examples.
  • the ETEC polypeptide construct can not only serve as antigen against ETEC but also serve as a protein carrier for polysaccharide antigens, such as C . jejuni capsule polysaccharide.
  • Example 1 Conjugation of ETEC polypeptides to C. jejuni capsule polysaccharide (CPS)
  • ETEC recombinant polypeptides or polypeptide constructs are conjugated to C . jejuni CPS.
  • the CPS can be derived from a number of C . jejuni strains.
  • any CPS of any C . jejuni strain is envisioned to be conjugated to ETEC recombinant polypeptide constructs.
  • Shigella LPS can be conjugated to ETEC recombinant polypeptide constructs.
  • the overall method of conjugating includes oxidizing C . jejuni CPS, for example, with NaIO 4 in sodium acetate (pH 4.0). Oxidized CPSs were desalted with a 5 kDa cutoff membrane by stirred ultrafiltration, which is subsequently lypholized. ETEC proteins are then added.
  • the stoichiometery protein to CPS can vary, however, a typical ratio is 1:2 protein to CPS by mass.
  • the concentration of components can be by any method. However, for example, polysaccharide concentration was determined by antrhone assay and protein concentration was determined. NaCNBH 3 is then added. The conjugates are subsequently desalted by ultrafiltration and lyophilized.
  • CPS or Shigella LPS
  • ETEC proteins and conjugates were analyzed, for example by SEC-HPLC.
  • Conjugates were also analyzed by SDS polyacrylaminde gel electrophoresis (PAGE) and Gel Code ® Blue (Pierce, Biotechnology, Inc, Lombard, IL) staining. Conjugates were detected by antibody-based assay using anti-CPS and anti-ETEC protein.
  • the CPS from the C. jejuni strain 81-176 was conjugated to ETEC recombinant polypeptide construct CfaE (class 5 ETEC adhesin) or to the recombinant polypeptide construct CfaE linked, via a polypeptide linker, to the major subunit CfaB.
  • jejuni capsule was purified from Campylobacter jejuni strain 81-176 (PG3208).
  • This mutant in which the gal T gene was insertionally inactivated by chloraphenicol cassette, lacks all ganglioside mimicry in its lipoligosaccharide (LOS) core.
  • LOS lipoligosaccharide
  • the cells were grown in porcine Brain-Heart Infusion (BHI) broth and sonicated to inactivate the cells.
  • the CPS was extracted by hot water/phenol method previously employed for the same organism ( Chen, et al., Carbohyd. Res. 243: 1034 (2008 )).
  • Cells were immersed in a water/phenol mixture (3:2 ratio by volume), which was heated to 67°C with stirring for 4 hours.
  • the suspension was cooled and separation of the mixture into two separate layers (the aqueous layer and the phenol layer) and extraction of the aqueous layer was performed.
  • the aqueous layer was removed and the phenol/water extraction was repeated on residue, to maximize the yield.
  • Aqueous layers from two extractions were pooled and boiled fro 1.5 hours with the aditon of acetic acid to a pH of 3.5.
  • the aqueous layer was dialyzed gainst running water for 2 days and concentrated using a Millipore concentrator cell with a 5 kDa cutoff membrane. Trace amounts of residual RNA were removed by digestion with benzonase enzyme at 90 u/ml in 50 mM Tris-CCl/1 mM MgCl 2 ,pH 8 overnight at 37°C.
  • Benzonase was removed from CPS, then desalted and concentrated using stirred ultrafiltration with 30 and 5 kDa MWCO disc membranes, respectively.
  • the isolated CPS was oxidized with adding 40 mg of CPS to 40 mM NaIO 4 in sodium acetate pH 4 in the dark at 4°C for 2 days. Oxidized CPS was desalted with 5kDa cutoff membrane by stirred ultrafiltration and was subsequently lyophilized.
  • the ETEC proteins Prior to conjugation the ETEC proteins, for example dscCfaE and dscCfaEB, were transferred to 0.1M borate buffer at pH 9.0. Oxidized CPS was added to each ETEC protein at a ratio of 1:2, protein to CPS by mass, and then NaCNBH 3 was added at 2 times mass equivalent to CPS. The reaction was incubated 1 day at room temperature and 6 days at 37°C in the dark with continuous stirring. The conjugates were desalted by stirred ultirafiltration with 30 kDa membrane and lyophilized. Conjugates of the CPS to dscCfaE and dscCfaEB was conducted by SEC-HPLC. and polyacrylamide gel (PAGE) (12.5%) electrophoresis.
  • PAGE polyacrylamide gel
  • FIG. 1 illustrates the basic recombinant construct design.
  • the construct design comprises one, or more ETEC major or minor fimbrial subunits or fragments of major fimbrial subunits, containing the donor strand, derived from the same ETEC fimbrial type, which are connected, via polypeptide linkers and stabilized by donor strand complementation.
  • the construct can contain a deletion of the N-terminal region of the N-terminal subunit. This feature prevents undesirable associations with other monomers or multimers.
  • the C-terminal subunit is connected to and stabilized by a donor ⁇ strand, connected to the subunit via a polypeptide linker, wherein the donor ⁇ strand is either derived from the adjacent subunit (i.e., homologous) or from a different subunit of the same fimbrial type (i.e., heterologous).
  • FIG. 2 illustrates the basic multipartite construct, wherein multiple constructs as in FIG. 1 , are connected forming a recombinant construct comprising two or more fimbrial types. As illustrated in FIG. 1 , major or minor subunits from the same fimbrial type are connected via a polypeptide linker sequence. In the multipartite construct, two or more constructs, as in FIG. 1 , are connected, via a linker polypeptide.
  • the first subunit is a major or minor ETEC fimbrial subunit.
  • Each additional subunit is connected to adjacent subunits via a polypeptide linker that enables rotary freedom of the molecular components.
  • the subunits are associated with and stabilized via a donor strand complementation from a C-terminally adjacent subunit via a donor ⁇ strand, connected via a linker polypeptide, to the C-terminus of the stabilized subunit.
  • subunits can contain a deletion of 14 to 18 amino acids from its N-terminal end.
  • specific constructs can be constructed with or without signal peptides of 18 to 22 amino acids and with or without histidine tags at the C-terminus.
  • Fimbrial types include, but are not limited to ETEC class 5a, 5b, 5c, CS3 and CS6.
  • a single construct can include subunits derived from any two or more of class 5a, 5b, 5c, CS3 and CS6 fimbrial types.
  • linker sequences can be utilized in connecting the individual subunits.
  • specific linkers include the tetrapeptide of SEQ ID No. 5.
  • Another example is a tri-glycine linker (i.e., G-G-G).
  • G-G-G tri-glycine linker
  • in cis donor strand complementation is used to stabilize adhesins and adhesin-pilin fusions for representative Class 5a, 5b, and 5c adhesins.
  • the contemplated composition is designed to enable as wide a range of coverage of ETEC strains as possible.
  • the contemplated composition and use is aimed at inducing immunogenic response against class 5a, 5b, 5c ETEC, as well as ETEC strains expressing CS3 or CS6 fimbrial components.
  • recombinant polypeptide ETEC constructs are conjugated to C. jejuni capsule polysaccharide (CPS).
  • C. jejuni capsule polysaccharide C. jejuni capsule polysaccharide
  • One or more of a number of ETEC recombinant constructs can be conjugated to one or more of a number of C. jejuni capsule polysaccharide structures.
  • Examples of Class 5 ETEC recombinant polypeptides are listed in Table 2. In Table 2, minor subunits are stabilized by connection, via a polypeptide linker, to associated major subunits. Alternatively, a 12-16 amino acid donor strand, derived from the associated major subunit is connected to the minor subunit via a polypeptide linker. These polypeptides can also be linked as per FIG. 1 and FIG.
  • the mature polypeptide sequence therefore, would be the full length minus the signal peptide.
  • 2 DNA sequence encodes mature protein.
  • the number and subunit refers to the N-terminal amino acids of length represented by the number from the subunit indicated that is connected at the C-terminus of the construct and is serving to stabilize the C-terminal construct.
  • dsc 14CsfA refers to the N-terminal 14 amino acids of CsfA connect to the C-terminus of the construct.
  • 2 Linkers polypeptides are GGG rather than DNKQ.
  • 3 Sequence in example contains a Leu-Glu-His 6 at the C-terminus.
  • ETEC adhesin ETEC adhesin
  • ETEC Class 5 fimbriae ETEC Class 5 fimbriae
  • an important feature of the recombinant constructs is the vastly improved stoichiometric representation of the minor subunit in order to enhance immune recognition of the minor subunit.
  • fimbrial subunits such as CfaE
  • stabilization of the adhesin is also important. Therefore, constructs are designed to express ETEC subunits stabilized from misfolding and degradation by donor strand complementation.
  • the donor ⁇ strand is provided by the major fimbrial subunit.
  • stabilization is provided by the N-terminal region of CfaB.
  • dscCfaE is folded into a native, ⁇ -sandwich conformation, consisting of two half-barrels, comprising the N-terminal adhesin domain (CfaEad) a short ⁇ -helical connector, and the C-terminal pilin domain (CfaEpd).
  • the molecule is functional in that it directly mediates MRHA of bovine and human erythrocytes, and generates neutralizing antibodies that act to inhibit MRHA and decorate the tips of CFA/I fimbriae on immunoelectron microscopy.
  • a fusion protein was engineered by genetic insertion of the coding sequence for mature major structural subunit of ETEC adhesin, such as CfaB, to the 3'-end of the minor subunit, such as CfaE.
  • This concept was disclosed in Savarino, U.S. Patent application (11/340,003, filed January 10, 2006 ).
  • This molecule contains all three domains of the CFA/I fimbriae (i.e., ad, pd, and major subunit) in a ratio of 1:1:1, rather than that found in native fimbriae (ca. 1:1:1000).
  • CfaE a Class 5a fimbrial adhesin
  • CsuD of CS14 fimbriae and CsfD of CS4 fimbriae are 80-81% identical with the other Class 5a minor subunits proteins adhesins CsuD of CS14 fimbriae and CsfD of CS4 fimbriae.
  • CsuD and CsfD share 94% identity. This is considerably higher than the average identity with other Class 5b and 5c fimbrial adhesins (mean 50% identity).
  • rabbit anti-dscCfaE serum cross-neutralizes CS4- and CS14-ETEC in the hemagglutination assay (HAI).
  • HAI hemagglutination assay
  • An embodiment includes anti-class 5 ETEC constructs based on the construct design illustrated in FIG. 1 , whereby the N-terminal subunit is an ETEC class 5 minor (i.e., adhesin) subunit, listed in Table 2, including CfaE, CsfD, CsuD, CooD, CsdD, CosD, CsbD and CotD, connected, via a polypeptide linker, to one or more ETEC major subunits, from the same ETEC class 5 type, listed in Table 2.
  • the polypeptide linker can be any of a number of polypeptide sizes. In a preferred embodiment, the linker is a tetrapeptide with the polypeptide sequence of SEQ ID No. 5.
  • the C-terminal class 5 subunit is connected to a donor ⁇ strand, derived from a homologous subunit and is typically 12-19 amino acids.
  • one or more major subunit can include a deletion of 12 to 16 amino acids from the N-terminal region of the subunit.
  • FIG. 1 utilizes the concepts disclosed in Savarino, U.S. Patent application (11/340,003, filed January 10, 2006 )), including donor strand complementation to provide stabilized class 5 ETEC adhesin. Due to the homology of ETEC class 5 minor subunits and major subunits, FIG. 1 further contemplates multiple constructs incorporating the fimbrial subunits of Table 2, or derivatives of these polypeptides or DNA sequences.
  • the construct design incorporates the donor strand complementation stabilization features of Savarino (U.S. Patent application (11/340,003, filed January 10, 2006 )), and furthers it by incorporating multiple major subunits, from a specific ETEC type, into a single adhesin-pilin construct.
  • multiple class 5b major subunits can be connected to a class 5b adhesin (i.e, minor subunit).
  • Embodiments include adhesin-pilin constructs containing Csb D (ETEC Class 5b fimbrial adhesin) and Cot D (ETEC Class 5c fimbrial adhesin). Examples, for illustration, of embodiments of adhesin-pilin ETEC class 5 adhesin-pilin constructs, representing Class 5a, 5b and 5c are shown in Table 3.
  • the fimbrial structures may function as polyadhesins rather than monadhesins ( Zavialov, et al., FEMS Microbiol. Rev. 31: 478-514 (2007 )).
  • assembly of ETEC CS6 and CS3 may be mediated by a donor strand complementation mediated process through association of a CS6 or CS3 subunit with the N-terminal donor strand region of an adjacent subunit. Additionally, protection against misfolding and proteolytic degradation may also be afforded through donor strand complementation.
  • CS6 fimbriae comprise CssA and CssB. Whereas the two CS3 major subunits show little to no variation in polypeptide sequences, modest variation in CS6 proteins is observed. For example, greater than 90% identity is found in CS6 protein CssA and greater than 95% identity is found in CssB allotypes. Both CS6 structural proteins exhibit a relatively low level of variation (i.e., greather than 90% amino acid conservation), with greater variation in CssA and the mutations randomly distributed along the CssA polypeptide.
  • Monomeric CS6 subunit assembly appears to be mediated by donor strands from adjacent CS6 subunits, as discussed above. It is hypothesized that interaction to form these stable structures is mediated by inter-subunit interaction through donor strand complementation. Donor strand complementation also affords protection against misfolding and proteolytic degradation. Therefore, in a preferred embodiment, multimeric CS6 constructs were developed to take advantage of these attributes of donor strand complementation. Additionally, multimeric expression provides more efficient manufacture over production of monomers.
  • a construct conjugated to C. jejuni comprises a multimeric CS6 with one or more of the CS6 subunits, CssA and CssB, or allelic variation or derivatives, with the construct design configuration illustrated in FIG. 1 .
  • the construct comprises a dimer of CssB and CssA with CssB N-terminal to CssA (i.e., CssB-CssA).
  • CS6 subunit association is stabilized by in cis donor ⁇ strand complementation.
  • Donor strand complementation is afforded by linking a CS6 subunit at its C-terminus, to the donor ⁇ strand region of another CS6 subunit, via a tetrapeptide linker.
  • the linker can be any of a number of polypeptide regions. However, in a preferred embodiment, the linker is either as in SEQ ID No. 5 or a triglyicine linker.
  • stabilization is provided by donor ⁇ strand, connected at its C-terminus, from a homologous or heterologous CS6 subunit.
  • Homologous subunit is defined as two subunits of the same form (e.g., CssA OR CssB). Heteterologous subunits are of different forms (e.g., one is derived from CssA the other from CssB.
  • the CS6 donor ⁇ strand is typically the N-terminal 14-16 amino acid region of CS6 subunit.
  • the recombinant protein can be constructed with or without hexahistidine affinity tags, which are typically on the C-terminus.
  • the N-terminal 14-16 amino acids of the N-terminal CS6 subunit is deleted.
  • "dsc B14 CssBA” would contain a heterologous donor strand (i.e., "dsc"), from CS6 CssB, inserted at the C-terminus of the construct.
  • the donor strand is 14 amino acids in length, as indicated by the "14.”
  • a constructed designated “ntd 15 dsc A CssBA” would contain a homologous donor strand at the C-terminus of the construct and also comprises a deletion of the N-terminal amino acid region (termed “ntd”).
  • constructs comprise one or more CS6 subunits with amino acid sequences sequences selected from the group consisting of SEQ ID No. 2 (CssA) or SEQ ID No. 4 (CssB), or derivatives of these polypeptides.
  • the DNA sequence for CssA is SEQ ID No. 1 and for CssB, SEQ ID No. 3.
  • the subunits are connected by a polypeptide linker sequences.
  • the linker is a tetrapeptide with the amino acid sequence of SEQ ID No. 5.
  • CS3 contains both CstG and CstH, in near equal amounts. Therefore, dimeric constructs were devised incorporating CstG and CstH, according to the template construct design of FIG. 1 .
  • the polypeptide linker can be any number of polypeptide regions, however, in a preferred embodiment, the linker is a tetrapeptide of the sequence of SEQ ID No. 5, or a triglycine (i.e., G-G-G).
  • the donor ⁇ strand region is the N-terminal 14 - 16 amino acids of the mature CstH or CstG protein.
  • the first 14 - 18 amino acids of the N-terminal region of the N-terminal most subunit is deleted to avoid undesirable associations.
  • the CS3 construct is a dimer.
  • the recombinant polypeptide construct can be configured as "dsc 16CstH CstG-(linker)-CstH".
  • the mature CstG polypeptide (SEQ ID No. 101) or full length polypeptide sequence (SEQ ID No. 87) is connected at its C-terminus to CstH polypeptide (SEQ ID No. 99), via a polypeptide linker.
  • the CstH polypeptide is connected, at its C-terminus, to a donor ⁇ strand region of 16 amino acids derived from CstH via a polypeptide linker.
  • multipartite fusion or multipartite fusion constructs are recombinant polypeptide constructs according to FIG. 2 .
  • ETEC fimbrial types are defined as fimbrial proteins derived from fimbriae of different strain ETEC types, as listed in Table 4 or 5, or deriviates of these polypeptides or DNA sequences.
  • the fimbrial type "CS3" comprises CstH and CstG.
  • the fimbrial type “CS6” comprises CssA and CssB.
  • the fimbrial types of Class 5 ETEC include the fimbrial types Class 5a, Class 5b and Class 5c.
  • major and/or minor subunits, derived from the same ETEC fimbrial type are connected, via polypeptide linkers, and stabilized by donor ⁇ strand complementation, as illustrated in FIG. 1 .
  • a multipartite fusion comprises one or more fimbrial subunits of the same fimbrial type, as in FIG. 1 , connected to one or more fimbrial subunits derived from a different fimbrial type as illustrated in FIG. 2 .
  • the multipartite fusion construct can include a deletion of the N-terminal region of one or more fimbrial subunits, but is preferably on the N-terminal most fimbrial subunit for a given ETEC fimbrial type, as illustrated in FIG. 2 . This feature prevents undesirable associations with other monomers or multimers.
  • the size of the deletion of the N-terminal region is 14 to 18 amino acids.
  • multipartite fusion constructs comprising Class 5 adhesins do not contain a deletion of the N-terminal region.
  • the C-terminal subunit for an ETEC fimbrial type, is connected to and stabilized by a donor ⁇ strand, connected to the subunit via a polypeptide linker, wherein the donor ⁇ strand is either that derived from the adjacent subunit (i.e., homologous) or from a different subunit of the same fimbrial type (i.e., heterologous).
  • the size of the N-terminal donor strand depends on the fimbrial type and subunit stabilized. In preferred embodiments, for class 5 fimbrial subunits, the donor ⁇ strand, derived from the N-terminal region of the class 5 subunit stabilized, is 12 to 16 amino acids.
  • the donor ⁇ strand is 14 to 16 amino acids.
  • the construct can contain a deletion of the N-terminal region of the N-terminal subunit. This feature prevents undesirable associations with other monomers or multimers.
  • the size of the deletion of the N-terminal region is 14 to 18 amino acids.
  • FIG. 2 multiple constructs as in FIG. 1 are connected forming a recombinant polypeptide construct comprising two or more ETEC fimbrial types.
  • one or more major or minor subunits derived from the same ETEC fimbrial type, are connected via polypeptide linkers and stabilized by donor strand complementation.
  • one or more glycine residues separates different ETEC fimbrial types, acting as a "swivel" means between the ETEC types.
  • the glycine residue due to its small, unbranched molecular characteristics, enables rotary freedom of the molecular components.
  • Subunits derived from the same fimbrial type (as in FIG.
  • the construct can contain an N-terminal deletion at the N-terminus of the entire construct as well as an additional deletion, of 14 to 18 amino acids, at the N-terminus of the first "internal" subunit that is of a different fimbrial type. This is illustrated in FIG. 2 .
  • the deletion serves to shorten the length between subunits, thus reducing the likelihood of misfolding and proteolytic cleavage.
  • a donor ⁇ strand, derived from a homologous or heterologous subunit is inserted at the C-terminus of the C-terminal CS6 or CS3 subunit.
  • the donor ⁇ strand derived from the N-terminal region of the class 5 subunit that is stabilized, is 12 to 16 amino acids.
  • CfaB is stabilized by a 14 amino acid donor ⁇ strand; CsfA by a 14 amino acid donor ⁇ strand; CsbA by a 15 amino acid donor ⁇ strand, CooA by a 14 amino acid donor ⁇ strand and CotA by a 14 amino acid donor ⁇ strand.
  • the donor ⁇ strand is 14 to 16 amino acids, with preferred embodiments of CS3 fimbrial subunits (i.e., CstH or CstG) stabilized by a 16 amino acid donor ⁇ strand derived from CstH or CstG; and CS6 fimbrial subunits (i.e., CssA or CssB) stabilized with a 16 amino acid donor ⁇ strand derived from CssA or CssB.
  • CssA or CssB CS6 fimbrial subunits
  • other donor ⁇ strand lengths are envisioned.
  • the linker contains the amino acid sequence of SEQ ID No. 5.
  • the linker is a tri-glycine linker.
  • the C-terminal end of the construct contains a histidine tag for purification of the construct.
  • compositions are constructed with the intent of eliciting anti-adhesive immune responses.
  • Class 5 multipartite fusions comprising Class 5 adhesin minor subunits are typically construct such that the adhesin (i.e., minor fimbrial subunit) is located at the N-terminus of the constructed with the minor fimbrial subunit linked at its C-terminus to one or more major subunits, followed at the terminal end of the construct with the donor 0-strand of the last major subunit.
  • adhesin i.e., minor fimbrial subunit
  • constructs comprising Class 5a adhesin CfaE tandemly linked at its C-terminus to one or more of CfaB (CFA/I major subunit), CsuA2 (CS 14 major subunit) and CsfA (CS4 major subunit); Class 5b adhesin CsbD tandemly linked at its C-terminus to one or more of CsbA (CS17 major subunit), which shares high identity to the CS19 pilin subunit CsdA, and CooA (CS1 major subunit), which shares high identity to the PCFO71 pilin subunit CosA; and Class 5c adhesin CotD tandemly linked at its C-terminus to CotA (CS2 major subunit).
  • ETEC multipartite fusion constructs are illustrated in Table 4 and 5.
  • constructs comprise any major or minor ETEC fimbrial subunit from Table 2 in multiple combinations, connected by linker polypeptides and stabilized from proteolytic degradation by donor strand complementation utilizing the design illustrated in FIG. 2 .
  • Table 2 lists the ETEC fimbrial subunits (major and minor subunits) than can be used and incorporated into the multipartite fusion construct design of FIG. 2 , which can then be conjugated to C. jejuni capsule polysaccharide or Shigella LPS. Any subunit, therefore, is combined with one or more other ETEC major subunits from any ETEC fimbrial phenotypic type, including Class 5a, 5b, 5c, CS3 and CS6.
  • the recombinant polypeptide construct motif comprises a whole or immunogenic fragment of a minor or major ETEC fimbrial subunit connected at its C-terminal end to a linker.
  • the linker is connected at its C-terminus to a whole major ETEC fimbrial subunit or a polypeptide donor strand of an ETEC major structural subunit, derived from the same fimbrial type.
  • the whole ETEC major subunit or donor strand polypeptide is then connected, via a linker at its C-terminal end, to one or more additional major structural fimbrial subunits, derived from the same fimbrial type, from Table 2.
  • constructs containing Class 5a, 5b and 5c pilin subunits are selected based on the relatedness of minor and major subunits within a particular ETEC fimbrial class (i.e., class 5a, 5b or 5c).
  • adhesin i.e., minor fimbrial subunit
  • a specific fimbrial type e.g., Class 5a
  • Further selection of subunits is guided and based on epidemiological study analysis in order to achieve optimum immunogenic coverage of ETEC strains. What C. jejuni capsule polysaccharide to conjugate is predicated primarily on epidemiological data suggesting pathogenicity of the strain providing the capsule polysaccharide. Although many C. jejuni strains exist, most are not pathogenic.
  • the linker polypeptide can comprise a four (4) amino acid sequence (tetrapeptide) or a tri-glycine. Also, as illustrated in FIG. 2 , the subunits are interconnected and stabilized by donor strand complementation, which is denoted by "dsc". In this nomenclature, the fimbrial subunit derivation is also indicated.
  • the N-terminal CS3 subunit "CstG” is connected, via a linker, to the CS3 subunit "CstH”, which is connected, via a linker, to a donor strand of 16 amino acids derived from "CstH.”
  • the N-terminal CS6 subunit "CssB” is connected, via a linker, as illustrated in FIG.
  • donor strand complementation of the "CssB" subunit is via a heterologous donor strand (i.e., derived from "CssA).
  • the examples contain a "G” (i.e., glycine) to provide a "swivel.”
  • G i.e., glycine
  • the N-terminal region of N-terminal CS6 subunit is deleted (delineated by "ntd") to avoid undesirable association with other CS6 subunits, as described above.
  • ntd the N-terminal region of N-terminal CS6 subunit
  • other combinations of major and minor subunits are contemplated utilizing the construct design illustrated in FIG. 2 and the fimbrial subunits of Table 2.
  • a six (6) histidine (i.e., His 6 ) tag is inserted.
  • constructs can be designed to include the histidine (i.e., His 6 ) tag or designed without this tag region. Additionally, some sequences contain the signal peptide (designated "spd” in Table 2 and 3) region. Constructs can be constructed with or without this region, as well, which may be added to improve manufacturing efficiency of the multipartite fusion construct.
  • Table 4 Fimbrial type SEQ ID No.
  • Subunits can be linked via either DNKQ or tri-glycine linker.
  • 3 (G) refers to glycine residue introduced to provide a "swivel.”
  • 4 "ntd” refers to N-terminal deletion (excised from mature protein) with extent of deletion (i.e., amino acids) indicated.
  • 5 “dsc” refers to span of N-terminal residues from donor ⁇ -strand, its amino acid length and its source.
  • Table 5 Fimbrial type SEQ ID No.
  • Subunits can be linked via either DNKQ or tri-glycine (GGG) linker.
  • DNKQ is used, except where indicated with (GGG).
  • 3 (G) refers to glycine residue introduced to provide a "swivel.”
  • 4 "spd” refers signal peptide. Number indicates number of amino acids.
  • 5 “ntd” refers to N-terminal deletion (excised from mature protein) with extent of deletion (i.e., amino acids) indicated.
  • 6 “dsc” refers to span of N-terminal residues from donor ⁇ -strand, its amino acid length and its source.
  • recombinant polypeptide constructs can contain a C-terminal toxin A subunit, such as cholera toxin A2 (CTA) to form a chimeric molecule.
  • CTA cholera toxin A2
  • a full-length or truncated CTA2 is connected to CS6 or CS3 multimeric recombinant polypeptide construct, such as a CS6 or CS3 dimer.
  • one or more CS3 fimbrial subunits are connected, as in FIG. 1 , via a polypeptide linker, preferably a tetrapeptide or triglycine.
  • the C-terminal most CS3 fimbrial subunit is then connected to a donor ⁇ strand, via a polypeptide linker.
  • the donor strand can be homologous or heterologous to the C-terminal fimbrial subunit.
  • the donor strand is then connected to a toxin fragment, such as CTA2.
  • the CS3-chimera example shown in Table 4 comprise the polypeptide sequence of SEQ ID No. 37, which is encoded by the DNA sequence of SEQ ID No. 36.
  • the N-terminal fimbrial subunit is CstG with a pelB leader (22 amino acids) connected at its N-terminal end (see FIG. 13).
  • the CstH is connected, via a polypeptide linker, to a 16 amino acid donor strand derived from the N-terminal 16 amino acids of CstH, which is connected to an A2 toxin fragment (i.e., CTA2).
  • LTB is also expressed.
  • LTB comprises the amino acid sequence of SEQ ID No. 39 and is encoded by the nucleotide sequence of SEQ ID No. 38. Once expressed, the LTB sequence would self assemble into a pentamer and associate, non-covalently, with the CS3-chimera to form a hetero-hexameric holotoxin-like structure.
  • CS6 toxin chimera examples are also illustrated in Table 5.
  • one or more CS6 fimbrial subunits are connected via a polypeptide linker, preferably a tetrapeptide or triglycine.
  • the C-terminal most CS6 fimbrial subunit is then connected to a donor ⁇ strand, via a polypeptide linker.
  • the donor strand can be homologous or heterologous to the C-terminal fimbrial subunit.
  • the donor strand is then connected to a toxin component (e.g., CTA2).
  • the chimera is co-expressed, with LTB, which self assembles into a pentamer to form a non-covalent association with the chimeric adhesion-toxoid fusion molecule.
  • the constructs are dimers of CS6 subunits, connected via a tetrapeptide linker, with the C-terminal fimbrial subunit connected, via a tetrapeptide linker to a donor ⁇ strand.
  • the donor ⁇ strand can be homologous or heterologous to the C-terminal most fimbrial subunit.
  • the donor strands are heterologous to the C-terminal fimbrial subunit.
  • the donor strand is then connected to a cholera toxin A2 (CTA2) subunit.
  • CTA2 cholera toxin A2
  • N-terminal subunit 43 which is encoded by the nucleotide sequence of SEQ ID Nos. 42.
  • the N-terminal subunit is CssA, with the N-terminal 15 amino acids of the mature CssA sequence deleted.
  • a pelB leader sequence 22 amino acids was also added, which is illustrated in FIG. 14.
  • C. jejuni capsule polysaccharide was extracted from C. jejuni strains selected based on their association with diarrheal disease.
  • CPS from bacteria was extracted by hot water-phenol extraction for 2 h at 70 °C.
  • the aqueous layer was dialyzed (1000 Da) against water followed by ultracentrifugation to separate the CPS from the LOS.
  • the supernatant material containing the CPS was subjected to sizeexclusion chromatography (Sephadex G50) for further purification to yield the intact CPSs.
  • Monosaccharide composition was performed using a procedure amenable to the alditol acetate method ( Chen, et al., Carbohydr. Res.
  • Table 6 Capsule type Polysaccharide structure HS1 HS44 ⁇ 4)-a-D-Galp-(1 ⁇ 2)-Gro-(1 ⁇ P ⁇ HS3 ⁇ 4)-[P ⁇ 3]-alpha-D-Gal-(1 ⁇ 3)-[P ⁇ 2/7]-6-d-alpha-D-ido-Hep-(1 ⁇ ; or ⁇ 4)-[P ⁇ 3]-alpha-D-Gal-(1 ⁇ 3)-[P ⁇ 2]-L-glyeero-alpha-D-ido-Hep-(1 ⁇ (where P represents O-methyl-phosphoramidate) HS4/13/64 ⁇ 3)-6-deoxy-beta-D-ido-Heptose-(1 ⁇ 4)-beta-D-GlcNAc-(1 ⁇ HS23/36 [ ⁇ 3)- ⁇ -D-Gal-(1 ⁇ 2)-6d- ⁇ -D-altro-Me-Hep-(1 ⁇ 3)
  • HS 5 contains a complex of variations of polysaccharides. These include the following structures:
  • Example 4 Induction of immune response by ETEC-Campylobacter capsule conjugates.
  • mice were immunized with escalating amounts of vaccines administered with alhydrogel ® (Sergent Adjuvants, Clifton, NJ). Mice received a total of two immunization at a 4-week interval.
  • mice immunized with HS36-CfaEB (10 ⁇ g, 60 ⁇ g) and HS36-CfaE (60 ⁇ g) exhibited significant levels of serum IgG antibodies specific against HS36 CPS (p ⁇ 0.05) (see FIG. 4 (A) ), comparared to pre-immune sera.
  • all groups of immunized animals had antibody levels that were significant increased (p ⁇ 0.05) compared to levels observed after only one immunization. This effect was not dose dependent at the vaccine doses tested.
  • the data shown in FIG. 4 illustrates that the conjugate vaccine comprising an ETEC adhesin-based carrier protein conjugated to a C. jejuni CPS is capable of inducing an immune response against both bacterial components, i.e., C. jejuni CPS and ETEC CfaE.
  • HAI hemagglutination inhibition assay
  • the HAI assays were conducted by evaluating samples from each animal. The samples were initially diluted 1:8, then diluted two-fold over a wide range of dilutions. Each serum dilution was incubated with an equal volume of CFA/I + ETEC bacteria (strain H10407), which further diluted the serum 1:2. The final lowest dilution tested dwas 1:16, which was the limit of detection (LOD). The pre-incubated mixture was subsequently mixed with bovine erythrocytes in the presence of 0.5% mannose in U-bottom 96-well plates. In the absence of anti-adhesive antibodies, the erythrocytes formed visible agglutinated "buttons" of cells.
  • HAI titer was the highest serial dilution that completely inhibited agglutination. If there was not detectable inhibition at the lowest serum dilution of 1:16, the samples were assigned a value of one-half of the detection limit (i.e., 8) for computational purposes.
  • mice immunized with HS36-CfaEB conjugate vaccine exhibited higher HAI titers.
  • significance difference was between mice immunized with an HS36-CfaEB (60 ⁇ g) and HS36-CfaE (10 ⁇ g).
  • FIG. 6 A polysaccharide constructed was synthesized as shown in FIG. 6 .
  • trityl group was selectively introduced to C-6.
  • benzoylation was performed on compound ( FIG. 7 , structure 2), however extensive migration observed during the introduction of MeOPN lead us to look for a more suitable protecting group. Therefore, allyl groups were selected to protect the C-2, C-3 and C-4 positions which were resistant to migration. Allyl groups were later deprotected with catalytic hydrogenolysis which proved to be compatible with the MeOPN modification.
  • an aminopentanyl linker was introduced to the anomeric position as a site for conjugation.
  • OMP 4-methoxyphenyl group
  • CAN cerium ammonium nitrate
  • the corresponding hemiacetal was then converted into trichloroacetimidate donor .
  • 5-Amino-N-phthalimido-pentanyl linker was then introduced with TMSOTf as activator at 0 °C.
  • Compound 5 ( FIG. 7 ) was collected with 65% as the ⁇ anomer and 29% as the ⁇ anomer. The removal of trityl group gave a free 6-hydroxyl group for modification.
  • galactose modified at the 6 carbon with O-methyl phosphoramidate is used to induce immunity against multiple C. jejuni strains, even those strains not expressing MeOPN-6-Gal.
  • the monosaccharide construct MeOPN-6-Gal was recognized by antibody against capsule polysaccharide isolated from HS23/36, conjugated to CRM 197 .
  • antibody against polysaccharide from HS4, conjugated to CRM 197 also elicited an equivalent response, as anti-HS23/36 CRM 197 conjugate, against MeOPN-6-Gal.
  • anti-HSl-CRM 197 also reacted to MeOPN-6-Gal, although to a somewhat less extent.
  • MeOPON-6-Gal The strong cross-reactivity with MeOPON-6-Gal exhibited against HS23/36 and HS4 antibody may be explained by the fact that MeOPN-6-Gal share epitopic structures with HS23/36 and HS4 capsule polysaccharides.
  • MeOPN group in both HS23/36 and HS4 is to a primary hydroxyl.
  • Example 6 Immunogenic composition against C. jejuni and enterotoxigenic Escherichia coli (ETEC) using a combined C. jejuni capsule / ETEC construct
  • a synthetic conjugate vaccine strategy can be developed to protect against multiple enteric pathogens. Most efforts at development of vaccines against bacterial enteric pathogens are limited to a specific pathogen. The ability to combine vaccines against multiple, antigenically variable pathogens in a single, multi-valent, injectable vaccine would greatly simplify approaches to prevent acquisition and transmission of these pathogens worldwide.
  • ETEC and C. jejuni are among the leading causes of bacterial diarrheal disease.
  • CJ has been causally linked to several serious sequelae including Guillain Barré Syndrome, irritable bowel syndrome, and reactive arthritis.
  • recent studies have indicated an association between CJ infections and malnutrition and growth stunting in young children in resource-limited settings.
  • conjugate vaccines containing CJ polysaccharide capsules that have proven to be immunogenic in multiple animal species and to confer protection against C. jejuni diarrhea in NHP.
  • the newer synthetic approach is based on recent data that the immunodominant epitope on CJ polysaccharide capsule conjugate vaccines is the MeOPN modification found on different sugars in different capsule types.
  • an immunogenic platform against both C. jejuni and ETEC can be created by linking synthetic MeOPN-sugars to different ETEC protein antigens.
  • the approach could also be extended to include Shigella lipopolysaccharides (synthetic or detoxified) conjugated to ETEC proteins.
  • Shigella lipopolysaccharides synthetic or detoxified conjugated to ETEC proteins.
  • this platform could form the basis of a multivalent vaccine against three major bacterial diarrheal pathogens. Conjugation can also serve as a protein carrier to enhance immunogenicity of the Campylobacter construct.
  • the overall method of conjugating includes oxidizing C. jejuni CPS, for example, with NaIO 4 in sodium acetate (pH 4.0). Oxidized CPSs were desalted with a 5 kDa cutoff membrane by stirred ultrafiltration, which is subsequently lypholized. ETEC proteins are then added.
  • the stoichiometery protein to CPS can vary, however, a typical ratio is 1:2 protein to CPS by mass.
  • the concentration of components can be by any method. However, for example, polysaccharide concentration was determined by antrhone assay and protein concentration was determined by Pierce 660 protein assay or the BCA assay. NaCNBH 3 is then added.
  • the conjugates can then be subsequently desalted by ultrafiltration and lyophilized.
  • CPS, ETEC proteins and conjugates were analyzed, for example by SEC-HPLC or by SDS polyacrylaminde gel electrophoresis (PAGE), or other methods.
  • the immunogenicity of CfaE-HS23/36 and CfaEB-HS23/36 conjugates was observed in mice, as well as induction of hemagglutination inhibition (HAI) titers against Cfal in mice.
  • the amino acid sequence of the dscCfaE construct used is SEQ ID No. 138 (nucleotide sequence is SEQ ID No. 139).
  • the dsc 19 CfaE amino acid sequence is SEQ ID No. 143 (nucleotide sequence is SEQ ID No. 142).
  • the amino acid sequence for dsc 19 CfaEB is SEQ ID No. 141 (nucleotide sequence is SEQ ID No. 140).
  • the CfaEB-HS23/36 conjugate was down-selected in order to proceed to studies in Aotus nancymaae.
  • This non-human primate (NHP) model was selected because it has been used as a diarrheal disease model for both ETEC and C. jejuni.
  • HEP non-human primate
  • the first such study which is the only one that has been completed, was a dose finding study followed by a C. jejuni challenge.
  • Animals that were immunized with CfaEB-HS23/36 intradermally with poly IC also showed an attack rate of 33% with a mean onset of 1.5 days and a duration of two days (45% efficacy).
  • the animals immunized subcutaneously with CfaEB-CPS showed between 67-100% efficacy against diarrheal disease.
  • the attack rate in the group immunized with 0.5 ug of the vaccine was 0 (0/5, with one animal that vomited after challenge being eliminated) and the attack rate in the group immunized with 3.5 ug of the vaccine was 20% (1/5, with one animal being eliminated due to diarrhea prior to challenge).
  • the single animal in this group that did develop diarrhea had a later onset of disease (day 9).
  • Fig. 9 Immune responses to CPS and to CfaE were measured by ELISA. Animals in groups 1, 2 and 3 displayed IgG responses to both antigens and and IgA response to CfaE. Hemagglutination inhibition (HAI) titers against ETEC strain H10407 expressing Cfal fimbriae were determined and are shown in Fig. 10 . The results indicate that HAI titers were detected in animals in groups 1, 2 and 3, with group 2 showing the highest titers.
  • HAI Hemagglutination inhibition
  • CssBA-HS3 vaccine is a recombinant form of the two subunits of CS6 that are fused together. This protein was conjugated to capsule from an HS3 strain by TEMPO oxidation. The conjugates were analyzed by SDS-PAGE and immunoblotting. Purified CssBA has a predicted Mr of 31.8 kDa. The conjugate of CssBA-HS3 CPS runs as two bands, one slightly smaller than CssBA and one that runs at approximately 60 kDa. The bands in the conjugate react with both anti-CssBA antiserum and antibodies to whole cells of HS3, indicating that polysaccharide has been conjugated to the protein.
  • mice were immunized subcutaneously with three doses of the vaccine given at 4 week intervals. Doses were 5 ug by weight or 25 ug by weight. Animals were bled at day 0 and two weeks after each immunization and the response to CssBA and to CPS were determined by ELISA. The results, shown in Fig. 11 , indicate that there was a robust response to both the protein and the polysaccharide at both doses.
  • LTB-HS4 vaccine LTB is the binding component of the heat labile enterotoxin of ETEC. Recombinant LTB, which is not toxic, was conjugated to the polysaccharide capsule of an HS4 strain by reductive amination. The conjugate was analyzed by immunoblotting as shown in Fig. 12 . Immunodetection with anti-LTB antiserum revealed a single band for LTB at approximately 10 kDa. The conjugate contained 4 major bands ranging from ⁇ 20kDa->75kDa that were reactive with both anti-LTB and anti-HS4 antiserum, indicating successful conjugation.
  • mice were immunized with three doses of either 5 or 25 ug (by weight) of the LTB-HS4 conjugate subcutaneously at 4 week intervals and the serum immune response was determined.
  • the results, shown in Fig. 12 indicate that there was a robust immune response to both the HS4 capsule and to LTB at both doses.
  • Shigella dysenteriae a human pathogen cause diseases such as diarrhea and bacilliary dysentaery: Shigella dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei are important enteropathogens Strains of Shigella spp. Express long-chain lipopolysaccharides. The chemical structures for many strains has been determined (see Liu, et al., FEMS Microbiol. Rev. 32: 627-653 (2008 )).
  • An object of this invention is a Shigella LPS-ETEC construct.
  • the construct comprises an ETEC construct, as the above examples, conjugated, to a Shigella LPS, as an alternative or in addition to C. jejuni capsule polysaccharide. It is envisioned that any of the Shigella spp. can be conjugated to the ETEC construct.
  • the Shigella flexneri 2a LPS is illustrated, as a potential LPS structure that can be conjugated to an ETEC construct, as follows:

Claims (15)

  1. Immunogenes Multi-Agens-Konstrukt, umfassend ein Campylobacter jejuni Kapselpolysaccharid, das mit einem Proteinträger konjugiert ist, wobei der Proteinträger ein rekombinantes Polypeptidkonstrukt von enterotoxigenem Escherichia coli (ETEC) umfasst,
    wobei das rekombinante ETEC-Polypeptidkonstrukt eine ETEC-Fimbrien-Minor- oder -Major-Untereinheit umfasst, die am C-Terminus mit dem N-Terminus einer oder mehrerer ETEC-Fimbrien-Major-Untereinheiten des gleichen ETEC-Fimbrientyps wie die ETEC-Fimbrien-Minor- oder -Major-Untereinheit über einen Polypeptidlinker verbunden ist,
    wobei jede der einen oder mehreren ETEC-Fimbrien-Major-Untereinheiten einen Donor-β-Strang an ihrem N-Terminus enthält und jede der ETEC-Fimbrien- Major-Untereinheiten über einen Polypeptidlinker miteinander verbunden ist,
    wobei der C-Terminus der einen oder mehreren ETEC-Fimbrien-Major-Untereinheiten über einen Linker mit einem Donor-β-Strang einer ETEC-Fimbrien-Major-Untereinheit des gleichen ETEC-Fimbrientyps wie die eine oder mehreren ETEC-Fimbrien-Major-Untereinheiten verbunden ist,
    wobei das rekombinante ETEC-Polypeptidkonstrukt einen C-terminalen Histidin-Tag am C-Terminus enthalten kann, und
    wobei die ETEC-Fimbrien- Minor- oder -Major-Untereinheiten von ETEC-Stämmen abgeleitet sind, die aus der Gruppe ausgewählt sind, die aus Klasse 5, CS3 und CS6 besteht, wobei die ETEC-Fimbrien-Minor-Untereinheit aus der Gruppe ausgewählt ist, die aus CfaE, CsfD, CsuD, CooD, CsbD, CosD, CsdD, CotD besteht und wobei die Major-Untereinheit aus der Gruppe ausgewählt ist, die aus CfaB, CsfA, CsuA2, CooA, CsdA, CosA, CsbA, CotA, CstG, CstH, CssA und CssB besteht.
  2. Immunogenes Multi-Agens-Konstrukt nach Anspruch 1, wobei der Proteinträger ein oder mehrere zusätzliche rekombinante ETEC-Polypeptidkonstrukte umfasst, wobei jedes der einen oder mehreren zusätzlichen rekombinanten ETEC-Polypeptidkonstrukte ETEC-Fimbrien-Untereinheiten eines anderen ETEC-Fimbrientyps als jedes der anderen Konstrukte enthält.
  3. Immunogenes Multi-Agens-Konstrukt nach Anspruch 1 oder 2, wobei das rekombinante Escherichia coli-Polypeptidkonstrukt eine Aminosäuresequenz umfasst, die ausgewählt ist aus der Gruppe bestehend aus SEQ ID Nr. 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 102, 103, 105, 107, 109, 112, 114, 138, 141 und 143.
  4. Immunogenes Multi-Agens-Konstrukt nach Anspruch 1 oder 2, wobei das rekombinante Escherichia coli-Polypeptidkonstrukt durch die Nukleotidsequenz der SEQ ID Nr. 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 40, 42, 101, 104, 106, 108, 110, 111, 113, 139, 140 und 142 codiert wird.
  5. Immunogenes Multi-Agens-Konstrukt nach Anspruch 1 oder 2, wobei das Campylobacter jejuni Polysaccharid eine sich wiederholende Trisaccharid-Struktur mit der Formel ist, ausgewählt aus der Gruppe bestehend aus:
    [→3)-α-D-Gal-(1→2)-6d-α-D-altro-Me-Hep-(1→3)-β-D-GlcNAc-(1→]n ;
    [→3)-β-6-Deoxy-D-ido-Heptose (1→4)-β-D-GlcNAc-(1→]n ;
    [→3)-α-Araf-(1→3)-6-d-α-gulo-Hepp-(1→]n ;
    [→3-β-GalpNAc-(1→]n ;
    Figure imgb0021
    Figure imgb0022
    Figure imgb0023
    Figure imgb0024
    [→2)-D-glycero-α-D-manno-Hepp-(1-4)-α-D-GIcp-(1→]n ;
    Figure imgb0025
    [→3)-L-beta-D-ido-Hep-(1->4)-beta-D-Glc-(1→]n , mit nicht stöchiometrischer Substitution von O-Methyl-Phosphoramidat an Position 2 der L-Glycero-beta-D-ido-Heptose;
    [→3)-6d-beta-D-ido-Hep-(1->4)-beta-D-Glc-(1→]n , abgeleitet von HS13, mit nicht stöchiometrischer Substitution von O-Methyl-Phosphoramidat an Position 2 oder/und 7 der 6-Desoxy-beta-D-ido-Heptose;
    [→3)-L-beta-D-ido-Hep-(1->4)-beta-D-Glc-(1→]n ;
    [→3)-L-alpha-D-ido-Hep-(1->4)-alpha-Gal-(1→]n , mit nicht stöchiometrischer Substitution von O-Methylphosphoramidat an Position 2 der 6-Desoxy-alpha-D-ido-Heptose;
    Figure imgb0026
    Figure imgb0027
    Figure imgb0028
    Figure imgb0029
     und
    [→3)-6d-alpha-D-ido-Hep-(1->4)-alpha-Gal-(1→]n , abgeleitet von HS3, HS13 und HS50 mit nicht stöchiometrischer Substitution von O-Methylphosphoramidat an Position 2 von L-Glycero-alpha-D-ido-heptose, wobei "n" 1 bis 100 ist, und
    wobei das Shigella-Lipopolysaccharid die Struktur hat:
    Figure imgb0030
    Figure imgb0031
  6. Immunogenes Multi-Agens-Konstrukt nach Anspruch 1 oder 2, wobei der Donor-β-Strang 12 bis 16 Aminosäuren enthält, wobei der N-Terminus der Minor- oder Major-Untereinheit ein Signalpeptid mit 18 bis 22 Aminosäuren enthält, wobei der N-Terminus der Minor- oder Major-Untereinheit ein Signalpeptid mit 18 bis 22 Aminosäuren enthält, wobei die Aminosäuresequenz des Polypeptid-Linkers die Aminosäuresequenz von SEQ ID NO. 5 oder ein Triglycin ist, und wobei eine oder mehrere Major-Untereinheiten eine Deletion der 14 bis 18 N-terminalen Aminosäuren enthalten.
  7. Immunogenes Multi-Agens-Konstrukt nach Anspruch 1 oder 2, wobei die Aminosäuresequenz der Escherichia coli Fimbrien-Minor-Untereinheit ausgewählt ist aus der Gruppe bestehend aus SEQ ID Nrn. 45, 46, 51, 52, 57, 58, 65, 71, 75, 79, 83, 88, 90, 93, 95 und 97 besteht, und wobei die Aminosäuresequenz der Escherichia coli Fimbrien-Major-Untereinheit Gruppe ausgewählt ist, die aus den SEQ ID Nrn. 2, 4, 48, 49, 54, 55, 60, 61, 63, 67, 69, 73, 77, 81, 85, 87, 89, 91, 92, 94, 96, 98, 99, 101, 135 und 137 besteht.
  8. Immunogenes Multi-Agens-Konstrukt nach Anspruch 1 oder 2 zur Verwendung in einem Verfahren zur Induzierung einer Immunantwort gegen C. jejuni Stämme, wobei das Verfahren eine Immunantwort gegen eine oder mehrere Enterobakterien, ausgewählt aus der Gruppe bestehend aus C. jejuni Stämmen und Escherichia coli, induziert, wobei das Verfahren die Schritte umfasst:
    a. Verabreichung des immunogenen Mehrfachwirkstoffkonstrukts nach Anspruch 1 oder 2 in einem Dosisbereich von 0,1 µg bis 10 mg pro Dosis;
    b. Verabreichung einer Verstärkungsdosis der Kapselpolysaccharidzusammensetzung in einem Dosisbereich von 0,1 µg bis 10 mg pro Dosis.
  9. Immunogenes Multi-Agens-Konstrukt zur Verwendung gemäß Anspruch 8, wobei das Campylobacter jejuni Kapselpolysaccharid die Polysaccharidstrukturen von Anspruch 5 umfasst.
  10. Immunogenes Multi-Agens-Konstrukt zur Verwendung gemäß Anspruch 8, wobei die Escherichia coli Fimbrien-Minor-Untereinheit ausgewählt ist aus der Gruppe bestehend aus SEQ ID Nrn. 45, 46, 51, 52, 57, 58, 65, 71, 75, 79, 83, 88, 90, 93, 95 und 97 besteht, und wobei die Aminosäuresequenz der Escherichia coli Fimbrien-Major-Untereinheit aus der Gruppe ausgewählt ist, die aus den SEQ ID Nrn. 2, 4, 48, 49, 54, 55, 60, 61, 63, 67, 69, 73, 77, 81, 85, 87, 89, 91, 92, 94, 96, 98, 99, 101, 135 und 137 besteht.
  11. Immunogenes Multi-Agens-Konstrukt zur Verwendung gemäß Anspruch 8, wobei das rekombinante Escherichia coli Polypeptidkonstrukt eine Aminosäuresequenz umfasst, die aus der Gruppe ausgewählt ist, die aus SEQ ID Nr. 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 41, 43, 102, 103, 105, 107, 109, 112, 114, 138, 141 und 143 besteht.
  12. Immunogenes Multi-Agens-Konstrukt zur Verwendung gemäß Anspruch 8, wobei das rekombinante Escherichia coli Polypeptidkonstrukt durch die Nukleotidsequenz der SEQ ID Nr. 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 40, 42, 101, 104, 106, 108, 110, 111, 113, 139, 140 und 142 kodiert wird.
  13. Immunogenes Multi-Agens-Konstrukt zur Verwendung gemäß Anspruch 8, wobei der Donor-β-Strang 12 bis 16 Aminosäuren enthält, wobei der N-Terminus der Minor- oder Major-Untereinheit ein Signalpeptid mit 18 bis 22 Aminosäuren enthält, wobei der N-Terminus der Minor- oder Major-Untereinheit ein Signalpeptid mit 18 bis 22 Aminosäuren enthält, wobei die Aminosäuresequenz des Polypeptid-Linkers die Aminosäuresequenz von SEQ ID No. 5 oder ein Triglycin ist, und wobei eine oder mehrere Major-Untereinheiten eine Deletion der 14 bis 18 N-terminalen Aminosäuren enthalten.
  14. Immunogenes Multi-Agens-Konstrukt nach Anspruch 1 oder 2, wobei das immunogene Multi-Agens-Konstrukt zusätzlich zu dem Campylobacter jejuni Kapselpolysaccharid Shigella Lipopolysaccharid umfasst.
  15. Immunogenes Multi-Agens-Konstrukt nach Anspruch 14, wobei das Shigella Lipopolysaccharid die Struktur hat:
    Figure imgb0032
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